It has been known that choline oxidase (E.C.1.1.3. 17) is produced by bacteria belonging to the genus Arthrobacter (Japanese Patent Publication Nos. 60-4716 and 60-46953), those belonging to the genus Alkaligenes (Japanese Patent Laid Open Publication No. 54-17182), those belonging to the genus Brevibacterium (Japanese Patent Laid Open Publication No. 53-66492), those belonging to the genus Corynebacterium (Japanese Patent Laid Open Publication No. 54-23191), mold fungi belonging to the genus Aspergillus (Japanese Patent Laid Open Publication No. 53-52687), those belonging to the genera Cylindrocarbon, Fusarium and Gibbella (Japanese Patent Laid Open Publication No. 54-35284), those belonging to the genus Penicillium (Japanese Patent Laid Open Publication No. 56-92787), actinomycetes belonging to the genus Streptomyces (Japanese Patent Laid Open Publication No. 57-132880) and the like.
The choline oxidase derived from bacteria belonging to the genus Arthrobacter has a molecular weight of 84,000 and thermostable up to about 40.degree. C. Its optimal reaction temperature is 40.degree. C. (Japanese Patent Publication No. 60-4716). The choline oxidase derived from bacteria belonging to the genus Alkaligenes has a molecular weight of 95,000, whose thermal stability is not higher than 37.degree. C. and the optimal reaction temperature is 40.degree. C. (Japanese Patent Laid Open Publication No. 54-17182). The choline oxidase derived from bacteria belonging to the genus Brevibacterium has a molecular weight of 97,000. It is thermostable at 45.degree. C. for 30 minutes and inactivated at 50.degree. C. Its optimal reaction temperature is within the range of from 20.degree. C. to 35.degree. C. (Japanese Patent Laid Open Publication No. 53-66492). The choline oxidase derived from mold fungi belonging to the genus Aspergillus is thermostable up to 35.degree. C. and rapidly inactivated at 40.degree. C. (Japanese Patent Laid Open Publication No. 53-52687). While no description has been found on thermal stability of the enzyme derived from bacteria belonging to the genus Corynebacterium, the optimal reaction temperature is within the range of from 20.degree. C. to 35.degree. C. While no description has been found on the thermal stability of the enzymes derived from mold fungi belonging to the genera Cylindrocarbon, Fusarium and Gibbella, the optimal reaction temperature of those enzymes is 35.degree. C. (Japanese Patent Laid Open Publication No. 54-35284). The enzyme derived from mold fungi belonging to the genus Penicillium is only stable at pH ranging from 7 to 9 at 25.degree. C. for one hour (Japanese Patent Laid Open Publication No. 56-92787). As the microorganism capable of producing the enzyme derived from the genus Streptomyces, only Streptomyces nigrifaciens has been known and the enzyme is thermostable up to 30.degree. C. and inactivated at 40.degree. C. Further, the growing temperature of the microorganism is 37.degree. C. or below (Japanese Patent Laid Open Publication No. 57-132880).
These known choline oxidases have molecular weights ranging from 84,000 to 97,000 and are thermostable up to 40.degree. C. or at 45.degree. C. for 30 minutes, but rapidly inactivated at 50.degree. C. The optimal reaction temperatures of all known choline oxidases are 40.degree. C. or below.
Namely, known choline oxidases have a drawback that their thermal stabilities are inferior.
On the other hands, in the case of using an enzyme for clinical tests, chemical analyses and the like, that having higher thermal stability and higher optimal reaction temperature can be used more advantageously. In general, an enzyme has a drawback that it is unstable in comparison with other chemical substances. The principal reason thereof has been considered to be thermal denaturation of an enzyme at a high temperature [Koso Kenkyu-ho (Methods for Studying Enzymes), edited by Shiro AKABORI, Vol. 1, pp. 245 to 246, Asakura Shoten, Japan, 1955]. The "advantageous use" means that an enzyme can be stored for a long period of time in the case of a long-term use, or an enzyme can stand a long-term use in the case of using it repeatedly in an immobilized state. Further, a higher optimal reaction temperature is also advantageous because the enzyme can be used at a higher temperature. In order to obtain choline oxidase which is excellent in thermal stability and has a high optimal reaction temperature, the present inventors have intensively investigated microorganisms capable of producing such enzyme. As the result, it has been found that actinomycetes which can grow at 45.degree. C. or higher produce the desired choline oxidase. Further, the present inventors have isolated and purified the enzyme and studied it to attain the present invention.